The mitochondrial DNA of Drosophila melanogaster has transcription units distributed over approximately 75% of its length. No RNA transcription has been detected from the A plus T-rich region which makes up the remaining 25% of the genome. Fine strucutre mapping of RNA-DNA hybrids will be used to obtain a detailed picture of sequence organization within the transcribed regions of the mitochondrial DNA. We will attempt to determine the polypeptides encoded by the mitochondrial genome using cell-free transcription and translation of cloned fragments of mitochondrial DNA. One half of the A plus T-rich region of the mitochondrial DNA shows a distinctive pattern of protection from in vivo cross-linking with me3-psoralen. We have shown that the pattern of protection from me3-psoralen cross-linking is a reflection of the association of the mitochondrial DNA with other molecules in vivo. This distinctive association occurs immediately adjacent to the origin of replication, suggesting that it is involved in either replication or in the association of the DNA with mitochondrial membranes. Procedures are being developed to isolate the mitochondrial chromatin intact so that this structure can be studied directly.